Licensing of IP3 receptors to evoke cytosolic calcium signals

Cells are specialized for different roles. This division of labour requires that cells talk to each other and sense their surroundings. These conversations occur across a barrier, the plasma membrane (PM), that surrounds every cell. Receptors in the PM are the cell's antennae: each detects a specific extracellular signal, changes shape, and then regulates intracellular signalling. The most widespread of these signalling pathways requires IP3 receptors (IP3Rs), which are channels that release calcium (Ca) from intracellular stores (ER). IP3 causes these channels to open, allowing Ca to leak rapidly from the ER, with two important consequences: Ca is delivered to the intracellular targets that it regulates and the loss of Ca from the ER stimulates opening of channels in the PM through which more Ca can flow into the cell. The key point is that IP3Rs are hubs through which extracellular signals evoke Ca signals and thereby cellular responses.

The building blocks of Ca signals evoked by IP3Rs are 'Ca puffs', which are brief Ca signals (lasting about a tenth of a second) that occur when a handful of clustered IP3Rs open together. A typical cell expresses about 10,000 IP3Rs, most of which move rapidly within the ER, but Ca puffs occur repeatedly at just a few fixed sites (involving no more than few hundred IP3Rs). By labelling endogenous IP3Rs with a tag that allows us to see them with a microscope while simultaneously observing Ca signals, we have shown that the only IP3Rs that evoke Ca puffs are immobile and parked beneath the PM. These observations established a hitherto unrecognised need for an additional level of IP3R regulation: namely what 'licenses' IP3Rs to respond?

Our recent work showed that a protein that is up-regulated in cells expressing a protein (KRas) commonly mutated in human cancer licenses IP3Rs. This protein, KRAP, tethers IP3Rs to the cellular skeleton (actin) beneath the PM and alongside the sites where Ca entry occurs. In cells without KRAP, all IP3Rs are mobile and IP3 fails to evoke Ca signals. Cells with extra KRAP have more immobile IP3Rs and IP3 evokes more Ca puffs and larger Ca signals. Hence, KRAP determines whether a cell can respond to extracellular stimuli with a Ca signal. This discovery is interesting because it links the subcellular geography of IP3Rs to their activity, and it suggests that licensing may be regulated by Ras proteins (mutated in many cancers) and by the actin-skeleton. The latter is intriguing because the actin-skeleton is dynamic and its activity is controlled by PIP2, which is also the molecule from which IP3 is made. Hence, PIP2 may have two roles in regulating IP3R activity: licensing them to respond by assembling and anchoring the actin-skeleton, and as the source of the IP3 that causes licensed IP3Rs to open. Licensing of IP3Rs by KRAP also suggests mechanisms by which loss of Ca from the ER may locally regulate Ca entry across the PM.

My proposal will use microscopes equipped to look at single proteins and tiny Ca signals in living cells to establish how KRAP licenses IP3Rs to respond. We use gene-editing methods to tag or manipulate key proteins to allow them to be seen at native expression levels as they regulate Ca signals. We will address three questions:

1. What is the composition and architecture of the complex where KRAP and IP3Rs interact?
2. What does KRAP do to IP3Rs to allow them ('license' them) to respond?
3. Does PIP2 have a dual role in IP3R regulation: licensing via regulation of actin and as a source of IP3?

We address fundamental issues in cell biology - namely, understanding the mechanisms by which a ubiquitous intracellular signalling pathway is regulated to deliver spatially organised signals. Our work also has potential, as we develop peptides to disrupt KRAP-IP3R interactions, to provide the first useful antagonists of IP3Rs that will be experimentally useful and may provide routes to new therapies.

IP3 receptors (IP3R) are intracellular Ca2+ channels and the hubs through which extracellular stimuli evoke Ca2+ signals. Most Ca2+ signals in animal cells are coordinated by IP3Rs, and the spatial organization of these signals determines cellular responses.

Ca2+ puffs, which are brief coordinated openings of a few clustered IP3Rs, are the building blocks of cytosolic Ca2+ signals. Cells express several thousand IP3Rs, most of which are mobile, yet all Ca2+ puffs occur at immobile IP3R clusters immediately beneath the plasma membrane (PM) and adjacent to the sites where store-operated Ca2+ entry occurs. Most IP3Rs do not respond to IP3. There is, therefore, a hitherto unexpected and additional level of IP3R regulation: namely they must be 'licensed' to respond.

Our recent results indicate that immobile IP3R clusters are tethered to F-actin by KRas-induced actin-binding protein (KRAP), which licenses them to respond. In cells lacking KRAP, there are very few immobile IP3Rs and IP3 fails to evoke Ca2+ puffs or (with more intense stimulation) global Ca2+ signals. Over-expressing KRAP increases the number of actin-tethered IP3R clusters, and IP3 evokes more Ca2+ puffs and larger global signals. Hence, KRAP licenses IP3Rs to respond, and endogenous levels of KRAP expression determine whether a cell will respond to IP3. These new findings implicate Ras and F-actin in IP3R licensing, and they suggest mechanisms for local regulation of store-operated Ca2+ entry. Since phosphatidylinositol 4,5-bisphosphate (PIP2) is both the source of IP3 and a major regulator of F-actin assembly and anchoring, we need to consider whether PIP2 regulates both licensing (via actin) and gating (via IP3).

We use optical microscopy with gene-editing to define the mechanisms by which KRAP licenses IP3Rs and its regulation. We address 3 questions:

1. How are IP3Rs tethered to actin by KRAP?
2. How does KRAP license IP3Rs?
3. Does PIP2 contribute to licensing of IP3Rs by KRAP?

Our work will advance understanding of one of the most widespread of intracellular signalling pathways: IP3-evoked Ca2+ signalling. Our proposal, focused on how tethering of IP3Rs to actin by KRAP licenses them to respond, integrates several areas of cell biology (notably, Ca2+ signalling, actin dynamics and Ras signalling). This fundamental question, allied with the ubiquitous importance of Ca2+ signalling and the contributions of IP3Rs to human pathologies will ensure that our work delivers impact. Our expected impacts across diverse fields within the academic community are described in Academic Beneficiaries.

Training
Staff are encouraged to develop the skills and experience required for independence. They engage fully with every aspect of the project from developing proposals, managing budgets, reviewing and developing research programmes, to preparing publications and presenting work. Staff apply state-of-the art techniques equipping them for work in the best labs. Staff gain experience of teaching by supervising project/PhD students, teaching practical classes and in a lecture on advanced techniques to final year students. All staff contribute fully to weekly lab meetings, where they present and critically evaluate work. In my absence, lab meetings are chaired by postdocs. A major impact is our proven ability to train staff equipped to meet future needs of industry, the public sector and academia. Our contributions to providing the UK and beyond with well-prepared scientists comes also from our public engagement activities. We are requesting appointment of a relatively senior research associate, for whom these benefits are likely to come at an important time as he/she prepares for independence, but they apply also the technical assistant and to the summer students that the RA is likely to supervise during the tenure of the appointment.

Public understanding and schools
My lab organises the Young Pharmas scheme, which seeks to inspire year-12 students, ensure that they appreciate the importance of creativity and critical evaluation in science, and the economic impact of pharmacology. Parents and teachers also gain exposure to these activities through the final poster session/guest lecture evening. Our interests in cell signalling feature in the Young Pharmas activities. We provide at least one placement for an undergraduate to gain research experience before deciding on postgraduate options. We request some funds (£740 over 3y) to partially support these activities.
Staff contribute to the Cambridge Science Festival with a demonstration of the interactions of drugs with receptors. We provide occasional visits to schools, providing practical experience of, for example, insect biology and microscopy.
We work with press offices to maximize the impact of our work by bringing it to more diverse audiences than our primary publications can reach. Two press releases from BBSRC described our recent work.
These activities encourage informed interest in science from students who have not yet finalised their career choices, and a more widespread appreciation of the importance of addressing fundamental questions in biology.

Health
Our work addresses fundamental questions in cell biology, but it is relevant to human medicine since mutant IP3Rs are associated with human disease. There are, however, no useful drugs that directly target IP3Rs in intact cells (experimentally or in the clinic). Our work will provide a deeper understanding of a hitherto unrecognised level of IP3R regulation - licensing by KRAP, which immediately suggests a new possible target for experimental and/or therapeutic intervention (the KRAP-IP3R interface, Sect 2.2).The impacts for clinical medicine are impossible to predict. We will actively engage with clinicians and the pharmaceutical industry to ensure that our findings are presented at an early stage to communities with direct interests in clinical development.